The goal of this project is to determine the mechanism(s) by which microencapsulation of donor islets and CTLA4-Ig treatment of recipient synergize to permit long-term survival of neonatal porcine islet xenografts in spontaneously diabetic NOD mice. There is a critical need for better insulin therapy to circumvent complications of insulin-dependent diabetes mellitus (IDDM). Our long-term goal is to develop techniques for transplantation of microencapsulated, xenogeneic islets to provide a durable, physiological source of insulin to diabetic patients. Microcapsules are the most promising technique for protecting donor islets from host immune destruction. Our capsules are biocompatible and xenogeneic pig islets in these capsules function>100 days in mice with chemically induced (SZN) diabetes, showing that factors required for long-term function of xenogeneic islets are available. Capsules protect xenografts by preventing host sensitization. NOD mice (with spontaneous diabetes) destroy encapsulated neonatal pig islets in 3 weeks. Biopsies reveal activated macrophages, immunoglobulins, IL-1, TNF, IFN-g, and IL-10. We postulate that NOD rejection is initiated by donor antigens that are shed from encapsulated islets and processed via the MHC class II pathway by host APC. These APC activate NOD CD4+ T-cells that mount a Th1 response, with donor islet destruction via cytokines. Survival of encapsulated pig islets in NODs is significantly prolonged (>100 days) by blocking NOD T-cell activation with CTLA4-Ig. We will test the hypothesis that CTLA4-Ig combined with microencapsulation alters the repertoire of NOD T-cells that recognize the cross-reactive antigens on neonatal pig islets. We will characterize in vitro NOD T-cell proliferative responses to porcine islets, using T-cell clones analyzed by flow cytometry, lymphokine ELISA and adoptive transfer in vivo to NOD-Scid mice. We will test the hypothesis that CTLA4-Ig plus microencapsulation induces donor-specific unresponsiveness (tolerance) by challenging with donor-specific, cryopreserved islets, skin, thyroid, and spleen cells. We will elucidate the cellular mechanism(s) responsible for CTLA4-Ig effects in this mode by substituting a mutant CTLA4-Ig which binds B7-1 selectively, or one which does not fix complement. We will test the hypothesis that preventing T-cell activation using a combination of reagents directed toward different immune response pathways will provide more robust protection against rejection, by treating NODs with anti-CD4 and anti-CD40L mAbs in conjunction with CTLA4-Ig.